Inducible System for Highly Efficient Production of Recombinant Adeno-Associated Virus (rAAV) Vectors

Abstract

Production of clinical grade gene therapy vectors for human trials remains a major hurdle in advancing cures for a number of otherwise incurable diseases. Disclosed herein are systems based on a stably trans formed insect cell lines harboring helper genes required for vector production. Specifically exemplified are system embodiments that take advantage of DNA regulatory elements from two unrelated viruses—AcMNPV and AA V2. System embodiments utilize rep and / or cap genes either stably transfected in cell lines or which are introduced into cells as an expression cassette in a vector. Rep and cap genes that are designed to remain silent until the cell is infected with a viral vector. Infection with viral initiates rescue / amplification of integrated AAV helper genes resulting in dramatic induction of the expression and assembly of rAAV. The arrangement of this specific embodiment provides high levels of Rep and Cap proteins in every cell thus improving rAAV yields by 10-fold. The described vectors are modular in design and may be utilized for the production of other multiprotein complexes.

Description

RELATED APPLICATIONS[0001]This application claims the benefit under 35 USC §119(e) of U.S. Ser. No. 61 / 165,994 filed Apr. 2, 2009 and U.S. Ser. No. 61 / 239,775 filed Sep. 3, 2009 which are incorporated herein by reference in their entirety.INTRODUCTION[0002]High production costs of clinical grade gene therapy vectors remain a major impediment preventing many research laboratories from entering the field. This is especially true for replication-deficient rAAV vectors which are produced, for the most part, by plasmid DNA co-transfection. Only recently, alternative scale-up production protocols such as those utilizing baculovirus expression vectors had been developed. Traditionally, BEVs have emerged as one of the most versatile systems for the protein production. They provide high yield combined with the posttranslational modifications of the proteins. In addition to basic protein production BEVs were utilized for more complicated tasks such as the synthesis of heterologous multiprotei...

AI Technical Summary

Benefits of technology

[0002]High production costs of clinical grade gene therapy vectors remain a major impediment preventing many research laboratories from entering the field. This is especially true for replication-deficient rAAV vectors which are produced, for the most part, by plasmid DNA co-transfection. Only recently, alternative scale-up production protocols such as those utilizing baculovirus expression vectors had been developed. Traditionally, BEVs have emerged as one of the most versatile systems for the protein production. They provide high yield combined with the posttranslational modifications of the proteins. In addition to basic protein production BEVs were utilized for more complicated tasks such as the synthesis of heterologous multiprotein complexes (1), production of a variety of virus-like particles, and for the assembly of gene therapy vehicles such as rAAV vectors (2). The latter strategy utilizes insect cells co-infected with three BEVs, a procedure potentially capable of manufacturing rAAV in ‘exa-scale’ format (3). While extremely promising, the original protocol had not been widely adopted due to several shortcomings including a requirement for the co-infection with three different helpers Bac-Rep, Bac-VP, and Bac-GOI (gene of interest flanked by AAV inverted terminal repeats). Only recently, H. Chen has reported a significantly improved system where rep and cap helper genes in the respective BEVs incorporated artificial introns (4).

Problems solved by technology

High production costs of clinical grade gene therapy vectors remain a major impediment preventing many research laboratories from entering the field.

While extremely promising, the original protocol had not been widely adopted due to several shortcomings including a requirement for the co-infection with three different helpers Bac-Rep, Bac-VP, and Bac-GOI (gene of interest flanked by AAV inverted terminal repeats).

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